Quantum dots are fluorescent semiconductor nanoparticles. The fluorescence of semiconductor nanocrystals significantly exceeds the brightness and photostability of conventional fluorophores, such as organic dyes, typically used in molecular biological and biochemical applications (Chan and Nie, Science 281, 2016-2018, 1998; Bruchez et al., Science 281, 2013-2016, 1998). The novel optical properties of semiconductor nanocrystals allow for ultrasensitive detection and quantification of semiconductor nanocrystals, and any molecules bound to them, with simple fluorescence illumination-detection sources (Chan and Nie, Science 281, 2016-2018, 1998; Michalet et al., Single Molecules 2, 261-276, 2001; Howarth et al., Proc. Natl. Acad. Sci. USA 102, 7583-7588, 2005; Agrawal et al., Anal. Chem. 78, 1061-1070, 2006; Alivisatos et al., Annual Review of Biomedical Engineering 7, 55-76, 53 plates, 2005; Vu et al., Nano Letters 5, 603-607, 2005). The ability to detect nanocrystals makes the use of semiconductor nanocrystals an ideal choice to replace typical tags (such as fluorescent tags) for diverse applications.
For example, as a fluorescent label for Western blots, semiconductor nanocrystals offer much higher sensitivity (picogram quantities) and more reliable quantification (100-fold linear concentration range) then traditional chemistries based on reactions catalyzed by horse radish peroxidase (Ornberg et al., Nature Methods 2, 2005; Bakalova et al., J. Am. Chem. Soc. 127, 9328-9329, 2005. In addition, by using semiconductor nanocrystals as an in situ immunohistochemical label, target biomolecules can be localized at the single molecule level in cells (Michalet et al., Single Molecules 2, 261-276, 2001; Grecco et al., Microscopy research and technique 65, 169-179, 2004; Sundara Rajan and Vu, Nano Letters 6, 2049-2059, 2006).
An indispensable feature of Western immunoblotting is its capability to fractionate and determine the size of specific proteins, thus making it a favored technique for routine protein analysis of complex biomixtures. Despite widespread use, Western immunoblotting faces significant limitations in detection sensitivity, making it difficult or impossible to use in situations requiring detection of trace proteins (less than 1 ng) or scarce biosamples (less than 105-106 cells). Detecting Western immunoblot signals at the level of single fluorescent tags would achieve the ultimate sensitivity limit in Western immunoblotting technology and allow even broader application of this invaluable technique. Other analytical techniques for protein detection have similar sensitivity problems.
Substantially-improved detection methods are needed to detect protein samples present at trace concentrations in complex, heterogeneous tissue and biofluid samples.